U.S. patent application number 13/422027 was filed with the patent office on 2012-10-18 for aligned fibrous materials with spatially varying fiber orientation and related methods.
This patent application is currently assigned to THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Brendon M. Baker, Jason Alan Burdick, Matthew B. Fisher, Robert L. Mauck, Amy M. Silverstein.
Application Number | 20120265300 13/422027 |
Document ID | / |
Family ID | 47007005 |
Filed Date | 2012-10-18 |
United States Patent
Application |
20120265300 |
Kind Code |
A1 |
Mauck; Robert L. ; et
al. |
October 18, 2012 |
Aligned Fibrous Materials With Spatially Varying Fiber Orientation
and Related Methods
Abstract
Provided are materials comprising layers of anisotropically
aligned fibers, the alignment of which fibers may be adjusted so as
to give rise to circumferentially-aligned fibers that replicate the
fiber alignment of native fibrous tissue, such as the meniscus or
the annulus fibrosis. Also provided are laminates formed from the
disclosed materials, as well as methods of fabricating the
disclosed materials and laminates.
Inventors: |
Mauck; Robert L.;
(Philadelphia, PA) ; Fisher; Matthew B.; (Media,
PA) ; Baker; Brendon M.; (Philadelphia, PA) ;
Silverstein; Amy M.; (Philadelphia, PA) ; Burdick;
Jason Alan; (Philadelphia, PA) |
Assignee: |
THE TRUSTEES OF THE UNIVERSITY OF
PENNSYLVANIA
Philadelphia
PA
|
Family ID: |
47007005 |
Appl. No.: |
13/422027 |
Filed: |
March 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2010/049111 |
Sep 16, 2010 |
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13422027 |
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61243660 |
Sep 18, 2009 |
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61594551 |
Feb 3, 2012 |
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Current U.S.
Class: |
623/14.12 ;
156/275.5; 264/465; 424/400; 424/93.1; 428/113; 428/114; 428/323;
428/338; 428/411.1 |
Current CPC
Class: |
Y10T 428/24124 20150115;
B32B 2250/02 20130101; Y10T 428/268 20150115; B29C 48/05 20190201;
A61F 2/3872 20130101; B32B 37/182 20130101; B32B 2535/00 20130101;
A61F 2/3094 20130101; Y10T 428/25 20150115; B32B 2310/0843
20130101; A61F 2/30965 20130101; B32B 5/26 20130101; Y10T 428/31504
20150401; B32B 2305/28 20130101; Y10T 428/24132 20150115; B32B 5/12
20130101; B82Y 30/00 20130101; D01F 1/10 20130101; D01D 5/003
20130101 |
Class at
Publication: |
623/14.12 ;
428/323; 428/113; 428/411.1; 428/338; 428/114; 156/275.5; 264/465;
424/400; 424/93.1 |
International
Class: |
B32B 5/16 20060101
B32B005/16; B32B 27/02 20060101 B32B027/02; D02G 3/22 20060101
D02G003/22; A61K 35/00 20060101 A61K035/00; B32B 37/14 20060101
B32B037/14; B29C 47/00 20060101 B29C047/00; A61K 9/00 20060101
A61K009/00; B32B 5/12 20060101 B32B005/12; A61F 2/38 20060101
A61F002/38 |
Goverment Interests
GOVERNMENT RIGHTS
[0002] This work was supported by the National Institutes of Health
(grant no. R01 AR056624) and by the Department of Veterans Affairs
(grant no. I01RX000174). The government has rights in this
invention.
Claims
1. A composition, comprising: a first layer comprising a first
population of polymeric fibers, at least some of the first
population of polymeric fibers comprising nanoscale bodies disposed
within; and a second layer comprising a second population of
polymeric fibers, the first and second layers being bonded together
at one or more locations.
2. The composition of claim 1, wherein the first population of
polymeric fibers comprises a polymer that is natural, synthetic,
biocompatible, biodegradable, non-biodegradable, bioabsorbable, or
any combination thereof.
3. The composition of claim 1, wherein the first layer, the second
layer, or both, further comprises a porogenic material.
4. The composition of claim 1, wherein at least some of the second
population of polymeric fibers comprise nanoscale bodies disposed
within.
5. The composition of claim 1, wherein a nanoscale body comprises
at least one cross-sectional dimension in the range of from about 1
nm to about 100 nm.
6. The composition of claim 5, wherein a nanoscale body comprises a
body having an aspect ratio in the range of from about 1 to about
100.
7. The composition of claim 5, wherein a nanoscale body comprises
an organic material, an inorganic material, or both.
8. The composition of claim 7, wherein the inorganic material
comprises a metal.
9. The composition of claim 8, wherein the metal comprises
gold.
10. The composition of claim 1, wherein at least a portion of the
first population of fibers are substantially aligned in a first
direction.
11. The composition of claim 10, wherein at least a portion of the
second population of fibers are substantially aligned in a second
direction.
12. The composition of claim 11, wherein the first and second
directions are non-parallel relative to one another.
13. The composition of claim 12, wherein at least a portion of the
second population of fibers is aligned perpendicular to at least a
portion of the first population of fibers.
14. The composition of claim 1, wherein at least a portion of the
first population of fibers, at least a portion of the second
population of fiber, or both, have an arcuate alignment.
15. The composition of claim 1, wherein the first population of
fibers, the second population of fibers, or both, has an
anisotropic alignment.
16. The composition of claim 1, wherein the at least some of the
first population of fibers differ from at least some of the second
population of fibers in composition, cross-sectional dimension, or
both.
17. The composition of claim 1, wherein at least one of the first
layer and the second layer has a population of cells disposed
thereon.
18. The composition of claim 17, wherein the first and second
layers have different populations of cells disposed thereon.
19. The composition of claim 1, wherein the first population of
fibers, the second population of fibers, or both, has an average
cross-sectional dimension in the range of from about 10 nm to about
10,000 nm.
20. The composition of claim 1, wherein bonding between the layers
is effected by mediated matrix-deposition with appositional
culture.
21. A method, comprising: irradiating a first fibrous layer
comprising a first population of polymeric fibers having a first
population of nanoscale bodies disposed within, the irradiating
being performed so as to bond at least a portion of the first layer
to a second fibrous layer comprising a second population of
polymeric fibers.
22. The method of claim 21, wherein the second fibrous layer
comprises a second population of nanoscale bodies disposed
within.
23. The method of claim 21, wherein a nanoscale body has at least
one cross-sectional dimension in the range of from about 1 nm to
about 100 nm.
24. The method of claim 21, further comprising disposing the first
population of nanoscale bodies within a polymeric fluid so as to
form a first mixture and electrospinning the first population of
polymeric fibers from the first mixture.
25. The method of claim 22, further comprising disposing the second
population of nanoscale bodies within a polymeric fluid so as to
form a second mixture and electrospinning the second population of
polymeric fibers from the second mixture.
26. The method of claim 21, wherein the first population of
polymeric fibers, the second population of fibers, or both,
comprises a material that is natural, synthetic, biocompatible,
biodegradable, non-biodegradable, biosorbable, or any combination
thereof.
27. The method of claim 21, wherein the first population of fibers,
the second population of fibers, or both, has an average
cross-sectional dimension in the range of from about 10 nm to about
10,000 nm.
28. The method of claim 21, wherein the first population of
polymeric fibers, the second population of polymeric fibers, or
both, comprises an anisotropic alignment of fibers.
29. The method of claim 28, wherein the first population of
polymeric fibers, the second population of polymeric fibers, or
both, comprises an arcuate alignment of fibers.
30. The method of claim 21, wherein at least some of the fibers in
the first layer have a different alignment than some of the fibers
of the second layer.
31. The method of claim 21, wherein at least some of the fibers in
the second layer are oriented essentially perpendicular to at least
some of the fibers of the first layer
32. A method, comprising: electrospinning, from a polymeric fluid,
a first population of polymeric fibers onto a first rotating
surface of a mandrel, the electrospinning being performed such that
at least a portion of the first population of polymeric fibers is
aligned on the first surface in an arcuate fashion.
33. The method of claim 32, wherein a spinneret containing the
polymeric fluid is oriented essentially perpendicular to the plane
of the first rotating surface of the mandrel.
34. The method of claim 32, wherein a spinneret containing the
polymeric fluid is oriented essentially parallel to an axis about
which the first rotating surface of the mandrel rotates.
35. The method of claim 32, wherein the polymeric fibers comprise a
polymer that is natural, synthetic, biocompatible, biodegradable,
non-biodegradable, biosorbable, or any combination thereof.
36. The method of claim 35, wherein the polymeric fibers comprise a
population of nanoscale bodies.
37. The method of claim 32, further comprising depositing a cell
onto the electrospun fiber.
38. The method of claim 32, wherein at least a portion of the first
rotating surface has a linear velocity during electrospinning of
between about 8 m/s and about 12 m/s.
39. The method of claim 32, further comprising electrospinning
polymeric fiber so as to form a body having at least one
cross-sectional dimension in the range of from about 10 micrometers
to about 1 cm.
40. The method of claim 32, wherein the first rotating surface of
the mandrel comprises a first conductive region and a second
conductive region separated by an insulating region disposed there
between.
41. The method of claim 40, wherein the electrospinning gives rise
to a plurality of polymeric fibers aligned radially relative to an
axis about which the first rotating surface of the mandrel
rotates.
42. A composition, comprising: a first layer comprising a first
population of polymeric fibers, the first population of polymeric
fibers having an anisotropic alignment that varies spatially within
the layer.
43. The composition of claim 42, wherein at least a portion of the
first population of polymeric fibers has an arcuate alignment.
44. The composition of claim 42, wherein at least a portion of the
first population of fibers has an average diameter in the range of
from 10 nm to about 10 micrometers.
45. The composition of claim 42, further comprising a population of
cells contacting the first population of fibers.
46. The composition of claim 42, wherein the first population of
polymeric fibers comprises a biocompatible polymer.
47. The composition of claim 42, further comprising a second
fibrous layer comprising a second population of fibers, the second
fibrous layer being bonded to the first fibrous layer.
48. The composition of claim 47, wherein the second fibrous layer
comprises a second population of fibers having an average diameter
in the range of from 10 nm to about 10 micrometers.
49. The composition of claim 47, wherein the second population of
fibers is aligned essentially parallel to one another.
50. The composition of claim 47, wherein the second population of
fibers has an arcuate alignment.
51. The composition of claim 47, wherein the second population of
fibers is aligned in a direction that differs from the alignment of
the first population of fibers.
52. The composition of claim 42, wherein the first population of
fibers comprises a population of nanoscale bodies disposed
within.
53. The composition of claim 47, wherein the first layer, the
second layer, or both, further comprises a porogenic material.
54. The composition according to claim 1, the composition being
shaped to as to approximate at least a portion of a knee meniscus,
an annulus fibrosis, or any combination thereof.
55. The composition according to claim 42, the composition being
shaped to as to approximate at least a portion of a knee meniscus,
an annulus fibrosis, or any combination thereof the laminate.
Description
RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of
international application PCT/US2010/049111, "Artificial Meniscal
Implants," filed on Sep. 16, 2010, which international application
claims priority to U.S. Application 61/243,660, "Artificial
Meniscal Implants," filed on Sep. 18, 2009. The present application
also claims priority to U.S. Application 61/594,551, "Aligned
Fibrous Materials With Spatially Varying Fiber Orientation And
Related Methods," filed on Feb. 3, 2012. All of the foregoing
applications are incorporated herein by reference in their
entireties for any and all purposes.
TECHNICAL FIELD
[0003] The present disclosure relates to the fields of
biocompatible implant materials and to biocompatible polymer
fibers.
BACKGROUND
[0004] The menisci are crescent-shaped fibrocartilaginous tissues
that function to transmit and distribute loads between the femur
and tibia of the knee joint. As such, the meniscus experiences
complex loads, including tension, compression, and shear. Meniscus
function in tension arises from an organized
microstructure--bundles of highly aligned collagen circumnavigate
the tissue between insertion sites on the tibial plateau. These
aligned collagen bundles endow the tissue with mechanical
properties that are highly anisotropic, and highest in the primary
collagen orientation. Existing meniscus replacement materials,
however, lack this unique structure and organization.
[0005] It is also known that load bearing fibrocartilaginous
tissues of the musculoskeletal system, including the knee meniscus
and the annulus fibrosus (AF) of the intervertebral disc, are prone
to failure and have a limited reparative capacity once damaged.
Both tissues are ordered hierarchical laminates: the knee meniscus
has a preponderance of circumferential collagen bundles with
interspersed, perpendicularly directed, `tie` fibers, while the AF
consists of multiple alternating layers of oriented (+/-about
30.degree.) collagen fibers that form an angle-ply structure. The
mechanical function of both tissues arises at least in part from
this underlying fibrous architecture.
SUMMARY
[0006] To address engineering the meniscus and other fibrous
tissues, presented here are aligned nanofibrous scaffolds that can
recapitulate this mechanical anisotropy. In natural tissues, fibers
within the native tissue have a pronounced c-shaped, or otherwise
angled, macroscopic organization. To replicate this macroscopic
change in organization over the anatomic size of the meniscus,
presented here is an electrospinning method that collects organized
fibers on a spinning disc or other mandrel.
[0007] This disclosure also presents data concerning the structure
and mechanics of nanofibrous scaffolds collected using this novel
technique, as compared to compare to aligned scaffolds obtained
from a traditional electrospinning approach. Without being bound to
any particular theory, one may hypothesize that these
circumferentially aligned (CircAl) scaffolds would behave similarly
to linearly aligned (LinAl) scaffolds on short length scales, but
exhibit marked differences in mechanics as the length scale
increased.
[0008] This disclosure presents aligned nanofibrous scaffolds
(formable from a variety of polymers) that can mimic the order of
these native tissues, and direct cell and matrix organization with
in vitro culture. Also disclosed are constructed biologic
laminates, in which scaffold layers are fused with one another
through cell mediated matrix-deposition with appositional culture.
In some embodiments, the tensile characteristics of the scaffold
may replicate those of a mammalian knee menisus. Since the
materials of construction in some cases exhibit non-linear stress
responses to strain and/or are biodegrade or bioerode when
subjected to physiological fluids under physiological conditions,
and the scaffold may continue to provide tensile support during
this period of biodegradation or bioerosion over a range of strain
conditions, it is often useful to characterize the scaffold in
terms of these parameters. That is, in various embodiments, a
material (e.g., a scaffold) according to the present disclosure
exhibits an overall circumferential modulus that is in the range of
about 10 MPa to about 200 MPa, preferably at least about 20 MPa,
more preferably at least about 40 MPa, still more preferably at
least about 60 MPa, and most preferably at least about 80 MPa, at a
strain region of about 10%, and/or an overall circumferential
modulus in the range of about 5 to about 60 MPa, preferably at
least 10 MPa, more preferably at least 20 MPa, and most preferably
about 30-35 MPa, at a strain region of about 3%, and these
properties are either retained or developed when the scaffold is
subjected to physiological implant conditions for time sufficient
to allow cell infiltration and meniscal healing, during and after
which the components of the matrix are dissolved, bioeroded, or
biodegraded into the patient. Preferably the modulus of the
scaffold, after exposure to physiological fluids under
physiological conditions, retains at least about 60% of its value
after 7 days, and more preferably at least about 50% of its value
after 60 days. In order to retain these modulus levels this
invention also provides that the scaffolds have correspondingly,
proportionately higher initial values. Unless otherwise stated
herein, any reference to a specific target modulus is intended to
reflect an initial value (i.e., before biodegradation or bioerosion
and the changes in mechanical properties that develop as cells
infiltrate and deposit new, load-bearing extracellular matrix
within the scaffold substance). It should be understood that any
and all mechanical characterizations or properties of materials set
forth in international application PCT/US2010/049111 may apply to
the materials disclosed herein.
[0009] Clinical application of these materials may, in some cases,
benefit from implantation of already formed acellular
multi-lamellar constructs. A `spot-welding` method has been
previously described in which method individual layers are bound
together through local scaffold melting brought on by contacting at
least one of the layers with a heated probe. This approach creates
stable bi-layers, but can cause compression of the construct with
insertion of the heated probes or arrays of probes.
[0010] An example of spot-welded layers is shown in FIG. 7. That
figure illustrates insertion of a heated probe into two adjacent
layers so as to fuse them together. The number of spot welds (lower
left of figure) affects the mechanical properties of the final
material, but the macroscopic structure of the spot-welded layers
(lower left of figure) is affected by insertion of the heated
probe. Here is presented a new method for forming nanofibrous
laminates using light responsive materials, which materials may be
polymeric fibers having nanoscale bodies (e.g., gold nanorods)
disposed within. The nanoscale bodies effect controlled levels of
heat with exposure to near-infrared (IR) light, which in turn
allows for fusion of layers without physical contact from a probe
or other instrument. This lack of contact in turn allows for layer
fusion without the disruption of the layers' underlying structure
that may result from contacting the layers with thermal probes or
other implements.
[0011] In one embodiment, the present disclosure provides laminates
(which may, in some places, also be referred to as compositions),
the laminates comprising a first layer comprising a first
population of polymeric fibers, at least some of the first
population of polymeric fibers comprising nanoscale bodies disposed
within; and a second layer comprising a second population of
polymeric fibers, the first and second layers being bonded together
at one or more locations.
[0012] The present disclosure also provides methods, the methods
comprising irradiating a first fibrous layer comprising a first
population of polymeric fibers having a first population of
nanoscale bodies disposed within, the irradiating being performed
so as to bond at least a portion of the first layer to a second
fibrous layer comprising a second population of polymeric
fibers.
[0013] Also provided are methods, the methods comprising
electrospinning, from a polymeric fluid, a first population of
polymeric fibers onto a first rotating surface of a mandrel, the
electrospinning being performed such that at least a portion of the
first population of polymeric fibers is aligned on the first
surface in an arcuate (which may be characterized, in some cases,
as being circumferential) fashion.
[0014] Further provided are compositions, the compositions
comprising a first layer comprising a first population of polymeric
fibers, the first population of polymeric fibers having an
anisotropic alignment that varies spatially within the layer.
[0015] Additionally provided are biocompatible implants, the
implants comprising a quantity of a composition according to the
present disclosure, the quantity of material being shaped to as to
approximate at least a portion of a knee meniscus, an annulus
fibrosis, or any combination thereof.
[0016] Further provided are methods, the methods comprising seeding
a composition according to the present disclosure with a population
of cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The summary, as well as the following detailed description,
is further understood when read in conjunction with the appended
drawings. For the purpose of illustrating the disclosure, there are
shown in the drawings exemplary embodiments of the disclosure;
however, the disclosure is not limited to the specific methods,
compositions, and devices disclosed. In addition, the drawings are
not necessarily drawn to scale. In the drawings:
[0018] FIG. 1 presents a schematic of nanofibrous scaffold
containing AuNRs interspersed in PCL fibers (A). MSCs align on NRS
(B) and SEMs show no difference between PCL (C) and NRS (D), scale
bar: 10 .mu.m. Average stress-strain profiles for PCL and NRS (E).
A small decrease in both modulus (F) and yield stress (G) was noted
in NRS compared to PCL scaffolds (n=6, *p<0.05).
[0019] FIG. 2 presents a schematic of laminate construction and
testing (left) and quantification (right) of maximum interface
strength for 1 and 2 welds produced through heated probe or IR
laser exposure of NRS (n=6, *=p<0.05);
[0020] FIG. 3 presents A) Bright field images of fibers collected
on slides (4.times.). B) Plot of mean fiber angle as a function of
position from center of scaffold;
[0021] FIG. 4 presents fluorescent imaging of actin (green) and
nuclei (blue) for MSCs seeded on linearly aligned (A) and
circumferentially aligned (B) scaffolds (scale bar=100 .mu.m);
[0022] FIG. 5 presents A) Schematic of specimens taken for tensile
testing from circumferentially aligned (CA) mats. B) Modulus of CA
and linearly aligned specimens with varying radii (3 cm, 5 cm),
sample length (short, long) and region for strain analysis (center,
edge). (*p<0.05 between short and long groups, +p<0.05
between scaffold region). C) Representative strain plots for LinAl
and CircAl scaffolds with a central region strain of 3%;
[0023] FIG. 6 illustrates the annulus fibrosis and the meniscus,
two fibrous tissues;
[0024] FIG. 7 illustrates an existing method of forming a
multi-lamellar nanofibrous structure;
[0025] FIG. 8 illustrates a micrograph of an annulus fibrosis and
also an image of a meniscus replacement material;
[0026] FIG. 9 illustrates exemplary parameters used in an
experiment involving dispersion of gold nanorods in
poly-caprolactone polymer;
[0027] FIG. 10 illustrates an exemplary process for fabricating
multilamellar materials according to the present disclosure;
[0028] FIG. 11 illustrates exemplary results realized from
fabricating multilamellar materials according to the present
disclosure;
[0029] FIG. 12 illustrates caprolactone nanofiber morphology
without (left) and with (right) inclusion of gold nanorods within
the nanofibers;
[0030] FIG. 13 illustrates a less-magnified view of FIG. 12;
[0031] FIG. 14 illustrates the morphology of a fibrous material
made from polycaprolactone fibers after spot welding (left) and
nanorod-infrared welding (right);
[0032] FIG. 15 illustrates mechanical data obtained from testing
fibrous scaffolds without ("PCL") and with ("NRS") nanorods;
[0033] FIG. 16 illustrates interface strength achieved by materials
according to the present disclosure;
[0034] FIG. 17 presents cell viability and morphology results
obtained on fibrous polycaprolactone scaffolds without (PCL) and
with (NRS) nanorod incusion;
[0035] FIG. 18 depicts an exemplary method of fabricating anatomic
meniscus structures according to the present disclosure; and
[0036] FIG. 19 depicts (A) a schematic showing electrospinning of
fibers onto a rotating mandrel; (B) and (C) bright field images of
strips of angled fibers with magnified images showing local
alignment.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0037] The present disclosure may be understood more readily by
reference to the following detailed description taken in connection
with the accompanying figures and examples, which form a part of
this disclosure. It is to be understood that this disclosure is not
limited to the specific devices, methods, applications, conditions
or parameters described and/or shown herein, and that the
terminology used herein is for the purpose of describing particular
embodiments by way of example only and is not intended to be
limiting of the claims. Also, as used in the specification
including the appended claims, the singular forms "a," "an," and
"the" include the plural, and reference to a particular numerical
value includes at least that particular value, unless the context
clearly dictates otherwise.
[0038] The term "plurality", as used herein, means more than one.
When a range of values is expressed, another embodiment includes
from the one particular value and/or to the other particular value.
Similarly, when values are expressed as approximations, by use of
the antecedent "about," it will be understood that the particular
value forms another embodiment. All ranges are inclusive and
combinable. Any documents cited herein are incorporated herein by
reference in their entireties for any and all purposes. Further
information may be found in U.S. patent applications 61/243,660 and
61/255,542, both of which are incorporated herein in their
entireties for any and all purposes.
[0039] In a first embodiment, the present disclosure provides
laminates. The laminates suitably include a first layer comprising
a first population of polymeric fibers, with at least some of the
first population of polymeric fibers comprising nanoscale bodies
disposed within. The laminates suitably include a second layer
comprising a second population of polymeric fibers, and the first
and second layers are suitably bonded together at one or more
locations.
[0040] The first population of polymeric fibers may comprise
virtually any polymer. Polymers that are natural, synthetic,
biocompatible, biodegradable, non-biodegradable, bioabsorbable, or
any combination thereof are all suitable. It should be understood
that in some embodiments, e.g., when an electrospun material is
made of a single fiber (e.g. nanofiber), the fiber is folded
thereupon, hence can be viewed as a plurality of connected fibers.
It is to be understood that a more detailed reference to a
plurality of fibers is not intended to limit the scope of the
present disclosure to such particular case. Thus, unless otherwise
defined, any reference herein to a "plurality of fibers" applies
also to a single fiber and vice versa.
[0041] This disclosure is not limited by the thickness or shape of
the fibers generated and used. Accordingly, the cross-sections of
the fiber or fibers may be circular, oval, rectangular, square, or
any shape which can be defined by the spinneret. Similarly, the
fibers can have a cross-sectional dimension in the range of about 1
nm to about 10 microns, in the range of about 20 nm to about 1000
nm, in the range of about 100 nm to about 1000 nm, or in the range
of about 1 micron to about 10 microns.
[0042] Fibers may be polymer fibers having diameters typically
between 10 nm and 1000 nm. Exemplary sub-ranges contemplated by the
present disclosure include between 100 and 1000 nm between 100 and
800 nm, between 100 and 600 nm, and between 100 and 400 nm. Other
exemplary ranges include 10-100 nm, 10-200 nm and 10-500 nm. As
mentioned, the fibers of the present disclosure are preferably
generated by an electrospinning processes. In certain preferred
embodiments, the first population of fibers, the second population
of fibers, or both, has an average cross-sectional dimension in the
range of from about 10 nm to about 10,000 nm.
[0043] As described herein, the various fibers may comprise
materials that are natural, synthetic, biocompatible,
biodegradable, non-biodegradable, and/or biosorbable, and unless
specifically restricted to one or more of these categories, the
fibers may comprise materials from any one of these categories.
[0044] The phrase "synthetic polymer" refers to polymers that are
not found in nature, even if the polymers are made from naturally
occurring biomaterials. Examples include, but are not limited to,
aliphatic polyesters, poly(amino acids), copoly(ether-esters),
polyalkylenes oxalates, polyamides, tyrosine derived
polycarbonates, poly(iminocarbonates), polyorthoesters,
polyoxaesters, polyamidoesters, polyoxaesters containing amine
groups, poly(anhydrides), polyphosphazenes, polysiloxanes, and
combinations thereof.
[0045] Suitable synthetic polymers for use according to the present
disclosure may include biosynthetic polymers based on sequences
found in collagen, elastin, thrombin, fibronectin, starches,
poly(amino acid), poly(propylene fumarate), gelatin, alginate,
pectin, fibrin, oxidized cellulose, chitin, chitosan, tropoelastin,
hyaluronic acid, polyethylene, polyethylene terephthalate,
poly(tetrafluoroethylene), polycarbonate, polypropylene and
poly(vinyl alcohol), ribonucleic acids, deoxyribonucleic acids,
polypeptides, proteins, polysaccharides, polynucleotides and
combinations thereof.
[0046] The phrase "natural polymer" refers to polymers that are
naturally occurring. Non-limiting examples of such polymers
include, silk, collagen-based materials, chitosan, hyaluronic acid
and alginate.
[0047] The phrase "biocompatible polymer" refers to any polymer
(synthetic or natural) which when in contact with cells, tissues or
body or physiological fluid of an organism does not induce adverse
effects such as immunological reactions and/or rejections and the
like. It will be appreciated that a biocompatible polymer can also
be a biodegradable polymer.
[0048] The phrase "biodegradable polymer" refers to a synthetic or
natural polymer which can be degraded (i.e., broken down) in the
physiological environment such as by enzymes, microbes, or
proteins. Biodegradability depends on the availability of
degradation substrates (i.e., biological materials or portion
thereof which are part of the polymer), the presence of
biodegrading materials (e.g., microorganisms, enzymes, proteins)
and the availability of oxygen (for aerobic organisms,
microorganisms or portions thereof), carbon dioxide (for anaerobic
organisms, microorganisms or portions thereof) and/or other
nutrients. Aliphatic polyesters, poly(amino acids), polyalkylene
oxalates, polyamides, polyamido esters, poly(anhydrides),
poly(beta-amino esters), polycarbonates, polyethers,
polyorthoesters, polyphosphazenes, and combinations thereof are
considered biodegradable. More specific examples of biodegradable
polymers include, but are not limited to, collagen (e.g., Collagen
I or IV), fibrin, hyaluronic acid, polylactic acid (PLA),
polyglycolic acid (PGA), polycaprolactone (PCL),
poly(Lactide-co-Glycolide) (PLGA), polydioxanone (PDO),
trimethylene carbonate (TMC), polyethyleneglycol (PEG), Collagen,
PEG-DMA, alginate or alginic acid, chitosan polymers, or copolymers
or mixtures thereof.
[0049] The phrase "non-biodegradable polymer" refers to a synthetic
or natural polymer which is not degraded (i.e., broken down) in the
physiological environment. Examples of non-biodegradable polymers
include, but are not limited to, carbon, nylon, silicon, silk,
polyurethanes, polycarbonates, polyacrylonitriles, polyanilines,
polyvinyl carbazoles, polyvinyl chlorides, polyvinyl fluorides,
polyvinyl imidazoles, polyvinyl alcohols, polystyrenes and
poly(vinyl phenols), aliphatic polyesters, polyacrylates,
polymethacrylates, acyl-sutostituted cellulose acetates,
nonbiodegradable polyurethanes, polystyrenes, chlorosulphonated
polyolefins, polyethylene oxides, polytetrafluoroethylenes,
polydialkylsiloxanes, and shape-memory materials such as poly
(styrene-block-butadiene), copolymers or mixtures thereof.
[0050] The phrase "biosorbable" refers to those polymers which are
absorbed within the host body, either through a biodegradation
process, or by simple dissolution in aqueous or other body fluids.
Water soluble polymers, such as poly(ethylene oxide) are included
in this class of polymers.
[0051] It will be appreciated that more than one polymer may be
used to fabricate the scaffolds of the present disclosure. For
example, the scaffold may be fabricated from a co-polymer. The term
"co-polymer" as used herein, refers to a polymer of at least two
chemically distinct monomers. Non-limiting examples of co-polymers
which may be used to fabricate the scaffolds of the present
disclosure include, PLA-PEG, PEGT-PBT, PLA-PGA, PEG-PCL and
PCL-PLA. The use of copolymers or mixtures of polymers/copolymers
provides a flexible means of providing the required blend of
properties. In but one non-limiting example, functionalized
poly(.beta.-amino esters), which may be formed by the conjugate
addition of primary or secondary amines with diacrylates, can
provide a range of materials exhibiting a wide array of
advantageous properties for this purpose. Such materials are
described, for example, in Anderson, et al., "A Combinatorial
Library of Photocrosslinkable and Degradable Materials," Adv.
Materials, vol. 18 (19), 2006, which reference is incorporated by
reference in its entirety.
[0052] Additionally, individual polymers or co-polymers may be
physically mixed and co-spun through the same spinneret. Similarly,
according to this disclosure, a composition may be comprised of a
mixture of simultaneously or sequentially delivered polymers and/or
copolymers. This includes mixtures of at least two natural,
synthetic, biocompatible, biodegradable, non-biodegradable, and/or
biosorbable polymers and co-polymers.
[0053] Other embodiments of this disclosure provide that, where a
composition comprises two or more fibers, that each may have a
different biodegradation and/or biosorption profile in a
physiological fluid, said fluids including water, saline, simulated
body fluid, or synovial fluid.
[0054] Still other embodiments provide that the polymers,
co-polymers, or blends thereof may be photolytically active, such
that once electrospun, they may be made to crosslink on exposure to
light, thereby improving the tensile characteristics of the
scaffold, and increasing the diversity and range of properties
available. See for example, Tan, et al., J. Biomed Matl. Res., vol.
87 (4), 2008, pp. 1034-1043, which is incorporated by reference in
its entirety.
[0055] In some embodiments, the first layer, the second layer, or
both, further comprises a porogenic material. At least part of the
porogenic material may be present as fibers, particles, or any
combination thereof. As used herein, the term "porogen" refers to
sacrificial materials added during the production of a scaffold
(for example, during electrospinning) and subsequently removed,
whose purpose is to occupy space during the construction process,
such that their subsequent removal results in what amounts to
engineered porosity. In tissue engineering, materials such as
inorganic salt like sodium chloride, crystals of saccharose,
gelatin spheres or paraffin spheres are used to introduce
particulate porosity. In the present disclosure, the use of porogen
fibers provides, in some embodiments, porosity aligned with the
remaining fibers.
[0056] The second population of fibers may include a population of
nanoscale bodies disposed within. A nanoscale body may be organic,
inorganic (e.g., metallic). A nanoscale body may also be a
biological molecule, such as a growth factor, a protease, trypsin,
and the like. A variety of dopants may be present within (or on)
the fibers of the disclosed materials.
[0057] In one set of embodiments, these dopants include at least
one therapeutic compound or agent, capable of modifying cellular
activity. Similarly, agents that act to increase cell attachment,
cell spreading, cell proliferation, cell differentiation and/or
cell migration in the scaffold may also be incorporated into the
scaffold. Such agents can be biological agents such as an amino
acid, peptides, polypeptides, proteins, DNA, RNA, lipids and/or
proteoglycans.
[0058] These agents may also include growth factors [e.g., a
epidermal growth factor, a transforming growth factor-.alpha., a
basic fibroblast growth factor, a fibroblast growth factor-acidic,
a bone morphogenic protein, a fibroblast growth factor-basic,
erythropoietin, thrombopoietin, hepatocyte growth factor,
insulin-like growth factor-I, insulin-like growth factor-II,
Interferon-.beta., platelet-derived growth factor, a nerve growth
factor, a transforming growth factor, a tumor necrosis factor,
Vascular Endothelial Growth Factor, an angiopeptin, or a homolog or
combination thereof], cytokines [e.g., M-CSF, IL-1beta, IL-8,
beta-thromboglobulin, EMAP-II, G-CSF and IL-IO, or a homolog or
combination thereof], proteases [pepsin, low specificity
chymotrypsin, high specificity chymotrypsin, trypsin,
carboxypeptidases, aminopeptidases, proline-endopeptidase,
Staphylococcus aureus V8 protease, Proteinase K (PK), aspartic
protease, serine proteases, metalloproteases, ADAMTS 17,
tryptase-gamma, and matriptase-2, or a homolog or combination
thereof] and protease substrates.
[0059] Suitable proteins which can be used along with the present
disclosure include, but are not limited to, extracellular matrix
proteins [e.g., fibrinogen, collagen, fibronectin, vimentin,
microtubule-associated protein ID, Neurite outgrowth factor (NOF),
bacterial cellulose (BC), laminin and gelatin], cell adhesion
proteins [e.g., integrin, proteoglycan, glycosaminoglycan, laminin,
intercellular adhesion molecule (ICAM) 1, N-CAM, cadherin,
tenascin, gicerin, RGD peptide and nerve injury induced protein 2
(ninjurin2)].
[0060] Additionally and/or alternatively, the materials of the
present disclosure may comprise an antiproliferative agent (e.g.,
rapamycin, paclitaxel, tranilast, Atorvastatin and trapidil), an
immunosuppressant drug (e.g., sirolimus, tacrolimus and
Cyclosporine) and/or a non-thrombogenic or anti-adhesive substance
(e.g., tissue plasminogen activator, reteplase, TNK-tPA,
glycoprotein IIb/IIIa inhibitors, clopidogrel, aspirin, heparin and
low molecular weight heparins such as enoxiparin and
dalteparin).
[0061] Examples of immunosuppressive agents which can be used to
minimize immunosuppression include, but are not limited to,
methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,
chloroquine, hydroxychloroquine, sulfasalazine
(sulphasalazopyrine), gold salts, D-penicillamine, leflunomide,
azathioprine, anakinra, infliximab (REMICADE), etanercept,
TNF-.alpha., blockers, a biological agent that targets an
inflammatory cytokine, IL-1 receptor antagonists, and Non-Steroidal
Anti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but
are not limited to acetyl salicylic acid, choline magnesium
salicylate, diflunisal, magnesium salicylate, salsalate, sodium
salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen,
indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,
nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin,
acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.
[0062] Cytokines useful in the present disclosure include, but are
not limited to, cardiotrophin, stromal cell derived factor,
macrophage derived chemokine (MDC), melanoma growth stimulatory
activity (MGSA), macrophage inflammatory proteins 1 alpha (MOP-1
alpha, 2, 3 alpha, 3 beta, 4, and 5, IL-, 11-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TNF-.alpha.,
and TNF-.beta.. Immunoglobulins useful in the present disclosure
include but are not limited to, IgG, IgA, IgM, IgD, IgE, and
mixtures thereof. Some preferred growth factors include VEGF
(vascular endothelial growth factor), NGFs (nerve growth factors),
PFGF-AA, PDGF-BB, PDGF-AB, FGFb, FGFa, and BGF.
[0063] It should be understood that the disclosed materials may
include a single type of nanoscale body/dopant, or two or more
types of nanoscale bodies/dopants.
[0064] A nanoscale body suitably has at least one cross-sectional
dimension in the range of from about 1 nm to about 100 nm. A
cross-sectional dimension is a length, width, diameter, or
thickness. A nanoscale body suitably has an aspect ratio in the
range of from about 1 to about 100. For example, a nanorod having a
height of 10 nm and a diameter of 5 nm would be considered
suitable. A nanorod having a diameter in the range of from about 2
to about 4 nm and a length in the range of from about 7 nm to about
9 nm is considered an especially suitable nanoscale body,
particularly when the nanorod is made of gold.
[0065] One embodiment of the present disclosure provides for the
selection of materials for the fibers of sufficiently high modulus
such that as one of the fibers degrades, the scaffold retains the
required modulus, for example at least 20 MPa, preferably at least
40 MPa, more preferably at least 60 MPa, and more preferably at
least about 80 MPa, at a strain region of about 10%, and/or an
overall circumferential modulus in the range of about 5 to about 60
MPa, preferably at least 10 MPa, more preferably at least 20 MPa,
and most preferably at least about 30-35 MPa, at a strain region of
about 3%, as defined herein, for sufficient time, for example over
10-20 weeks, under physiological conditions.
[0066] In other embodiments, a first fiber comprises a material
characterized as having a yield strain at least about 1%,
preferably at least about 4%, more preferably at least about 8% and
most preferably at least 10%. This fiber may be biocompatible, but
may or may not be biodegradable, though it is preferably so.
Absolute tensile properties of this material may beless important
than are those of the second fiber, since it is the combination of
the moduli of the first and second fibers, especially as a function
of time of exposure to physiological conditions, that are
important, but to accomplish this, the modulus of the first fiber
material should be at least about 20 MPa at lower (3%) strain
levels. Higher values are preferred, for example, preferably at
least about 100 MPa, and most preferably at least about 200 MPa,
especially at higher (10%) strain levels. In one embodiment, the
first fiber material comprises poly(caprolactone). In other
embodiments, this first fiber comprises a poly(.beta.-amino ester)
or an acrylate terminated poly(.beta.-amino ester). Such materials
are described, for example, in Anderson, et al., "A Combinatorial
Library of Photocrosslinkable and Degradable Materials," Adv.
Materials, vol. 18 (19), 2006, this reference being incorporated by
reference in its entirety.
[0067] A second fiber may comprise a biodegradable material
characterized as having a modulus in the range from about 10 MPa to
about 500 MPa, when subjected to a strain in the range of from
about 1% to about 10% and measured in the direction of the fiber
alignment; in another embodiment, this modulus is in the range of
about 20 MPa to about 500 MPa; in another, this modulus is in the
range of about 300 MPa to about 500 MPa, especially at higher (10%)
strain levels. In another embodiment, the second fiber comprises
poly(glycolic acid). In yet another embodiment, the second fiber
comprises a blend of poly(caprolactone) and poly(glycolic acid). In
other embodiments, this second fiber also comprises a
poly(.beta.-amino ester) or an acrylate terminated
poly(.beta.-amino ester). Yield stress for this fiber material
should be at least 1%, preferably at least about 4%, and most
preferably at least about 8%.
[0068] One embodiment of the invention provides that the first and
second fibers are be biodegradable, and that the rates of
biodegradability of the two fibers are different, when subjected to
similar or the same physiological conditions. In one embodiment,
the second fiber biodegrades more quickly than the first. In this
embodiment, when taken together, the relative rates of
biodegradability (or biosorption or dissolution) of the first
fiber, the second fiber, and the porogen fiber can be considered
slow, medium, and fast. It is preferred that the relative lifetime
of the second fiber in vivo is sufficiently long so as to provide a
sustained basis for tissue regeneration--generally on the order of
weeks under physiological conditions. The relative lifetime of the
second fiber in vivo can be determined or approximated by measuring
tensile properties or weight loss of the scaffold under simulated
physiological conditions.
[0069] In one embodiment, the scaffolding contains a porogen fiber,
co-spun with the first and the second fibers, comprising an amount
in the of about 10 to about 80 weight percent based on the total
weight of electrospun fibers, preferably in the range of about 20
to about 60 weight percent, more preferably in the range of about
30 to about 60 weight percent, and most preferably in about 40-55
weight percent, all with respect to the total weight of electrospun
fibers. As used herein, the term "porogen" refers to sacrificial
materials added during the production of a scaffold (for example,
during electrospinning) and subsequently removed, whose purpose is
to occupy space during the construction process, such that their
subsequent removal results in what amounts to engineered porosity.
In tissue engineering, materials such as inorganic salt like sodium
chloride, crystals of saccharose, gelatin spheres or paraffin
spheres are used to introduce particulate porosity. In the present
invention, the use of porogen fibers provides porosity aligned with
the remaining fibers.
[0070] In another embodiment, this porogen fiber has been removed,
such that the resulting scaffold contains spacings defined by the
absence of this porogen material. This removal can be accomplished
in several ways, though the most usual way of doing so is by
selective dissolution. Preferably, this porogen fiber is capable of
selectively and substantially dissolving in physiological fluids,
such as water, saline, meniscal fluid, simulated body fluid, or
synovial fluid, such dissolution occurring within one hour,
preferably within 30 minutes, and most preferably within 10 minutes
of contact with the physiological fluid at ambient or physiological
conditions. In such circumstances, one non-limiting example, the
porogen fiber comprises poly(ethylene oxide). However, the means of
removal is not limited to use of physiological fluids. For example,
depending on the porogen, hydrocarbons or other organic solvents
may also be used (ex vivo). Also the removal can be accomplished
inside or outside the patient. One of the purposes of applying and
then removing this porogen fiber material is to provide spaces
within the matrix to expedite cellular ingress into the scaffold
matrix, whose fiber densities otherwise inhibit this incursion. For
this reason, one skilled in the art would appreciate that removing
the porogen material from the matrix before seeding with, for
example, cell populations, and implanting into the patient can be a
desirable scenario.
[0071] Other embodiments describe the relative proportion of the
first and second fiber. In one such embodiment, the first fiber
comprises an amount in the range of about 20% by weight to about
80% by weight, relative to the combined weight of the first and
second fiber. Other embodiments define the relative amount of the
first fiber to be in the range of about 40% by weight to about 60%
by weight, or about 50% by weight, each relative to the combined
weight of the first and second fiber. The specific ratio of the two
fibers will depend on the particular choice of fibers, and one
skilled in the art would be able to understand the most appropriate
ratio for a given set of fiber materials based on the teaching
herein.
[0072] The invention teaches that the electrospun fibers may
individually comprise the individual polymers or copolymers, or
blends of polymers or copolymers or both. Within the scaffold
and/or within the individual fibers, the first fiber material may
be present in the range of about 1 to about 80 weight percent, and
the second fiber may be present in the range of about 80 to about 1
weight percent, each with respect to the total weight of
electrospun fibers.
[0073] Together, the first and second fibers may form a scaffold
whose circumferential modulus in the range of about 10 MPa to about
100 MPa, preferably in the range of about 60 MPa to about 90 MPa,
more preferably in the range of about 70 MPa to about 85 MPa, most
preferably about 80 MPa. This is accomplished by combining the
fiber materials, applied either as individual fibers or co-spun as
blended materials. such that the weighted average of the materials
according to their individual moduli provide the target scaffold
circumferential modulus. One skilled in the art would be able to
measure and/or calculate the combined modulus as a function of such
a composite. In the simplest case, this relationship can be
characterized according to the Rule of Mixtures equation:
[0074] Modulus of the composite=.SIGMA.[(.phi..sub.x*(modulus of
material x)],
[0075] where .phi..sub.x represents the weight fraction of the
x.sup.th component (strictly speaking, the rule of mixtures deals
with volume fractions, but to a good approximation, and assuming
polymers of comparable densities are used, use of weight fractions
provides an equivalent means of characterization).
[0076] Tensile modulus is a property which is often defined in
terms relative to the total cross-sectional area of the fiber or
fiber bundle, or in this case, to the circumferential alignment of
fibers. So as to maintain internal consistency, as described
herein, whether the scaffold contains or has had removed the
porogen fiber, the moduli are calculated and described so as to
consider the cross-sectional area of the porogen fiber, but not to
consider the tensile properties of that porogen fiber. For example,
in but one non-limiting example, a mixture comprising 25% by weight
(of the total polymer weight) of a first polymer, having a modulus
of ca. 20 MPa, and 25% by weight a second fiber, having a modulus
of 300 MPa, and 50% by weight of a porogen fiber, having a modulus
of 100 MPa is described herein as having a composite modulus of 80
MPa for the composite (i.e., (25%.times.20 MPa)+(25%.times.300
MPa)+(50%.times.0 MPa)=(5+75+0)=80 MPa), and not 130 MPa (as would
result if the modulus of the scaffold retained the contribution of
the porogen; i.e., (25%.times.20 MPa)+(25%.times.300
MPa)+(50%.times.100 MPa)=(5+75+50)=130 MPa) or 160 MPa (as would
result if the cross-sectional area of the porogen were ignored;
i.e., (50%.times.20 MPa)+50%.times.300 MPa)=(10+150)=160 MPa. It
should be appreciated that this definition provides a more rigorous
requirement for tensile modulus for the scaffold than if the
tensile contribution of the porogen material had been considered or
if the cross sectional area of the porogen material had been
ignored.
[0077] Other embodiments of this invention lift the constraint that
the first fiber have a particular yield stress value, and allowing
the second fiber to have a modulus lower in value than described
above, replacing these requirements with one that the combination
of first and second fibers maintain a mean circumferential scaffold
modulus of at least about 40 MPa, preferably about 60 MPA, and most
preferably at least about 80 MPa, at higher (10%) strain levels,
when subjected to physiological fluids under physiological
conditions for times sufficient to allow for cell ingress and
proliferation, typically on the order of weeks. As described
earlier, it is highly desirable that the scaffold maintain a
minimum modulus during the time of this cell ingress and
proliferation, corresponding to healing.
[0078] It is also understood that electrospinning provides fibrous
solid bodies which contain a degree of porosity which can be
affected by the materials of construction--both fibers and other
incorporated materials--and the method of making. Accordingly,
certain embodiments of this invention describe porous solids whose
void volumes are on the order of about 5 to 99 volume percent;
other embodiments describe porous solids with void volumes at the
lower end of this range, e.g., in the range from about 5 volume
percent to about 25 volume percent; still other embodiments
describe porous solids with void volumes in the middle of this
range, e.g., in the range from about 25 volume percent to about 75
volume percent; and still other embodiments describe solids with
void volumes at the high end of this range, e.g., in the range from
about 50 volume percent to about 95 volume percent. Other exemplary
sub-ranges contemplated by the present invention include the range
of about 80 to about 99 volume percent, the range of about 85 to
about 95 volume percent, and the range of about 90 to about 95
volume percent.
[0079] In the disclosed compositions, at least a portion of the
first population of fibers are suitably substantially aligned in a
first direction. It should be understood that not all of the first
population of fibers need be aligned in this first direction, which
first direction may be characterized as being about circumferential
to a hypothetical central axis, as illustrated in, e.g., FIG. 3b,
FIG. 4b, and FIG. 19. It is preferable that more than 50% of the
fibers be aligned in this first direction, but 50% should not be
understood as being a particular threshold. Similarly, at least a
portion of the second population of fibers are substantially
aligned in a second direction. The first a second directions may be
parallel to one another, although parallel directions are not a
requirement. In some embodiments, at least a portion of the second
population of fibers is aligned perpendicular to at least a portion
of the first population of fibers. In this way, the perpendicular
fibers may act as tie fibers in an artificial meniscus
material.
[0080] In some preferred embodiments, at least a portion of the
first population of fibers, at least a portion of the second
population of fiber, or both, have an arcuate alignment. Arcuate
should be understood as referring to fibers that are curved (as
opposed to being straight). The curve may be a circular or
circumferential one, although other curves (e.g., elliptical or
other curves that are not based on a constant radius) are also
suitable.
[0081] Illustrative fibers are shown in FIG. 3, which shows
linearly aligned fibers (FIG. 3A) and circumferentially-aligned
fibers (FIG. 3B). FIG. 3C illustrates the mean fiber angle
(degrees) as a function of the fiber's position from the center of
the mandrel. Data are shown for linearly-aligned fibers (which have
an unchanging fiber angle) and for circularly-aligned fibers (which
have an angle that changes as a function of the fiber's distance
from the center of the mandrel).
[0082] The first population of fibers, the second population of
fibers, or both, may have an anisotropic alignment. In some
embodiments, the compositions have a fiber alignment that varies
spatially within the body of the composition. This is described
below in additional detail.
[0083] At least some of the first population of fibers may differ
from at least some of the second population of fibers in
composition, cross-sectional dimension, or both. For example, some
of the first population of fibers may have an average diameter of
about 100 nm, and some of the second population of fibers may have
an average diameter of about 150 nm. The first population may be
formed from a first biocompatible polymer, and the second
population may be formed from a second, different biocompatible
polymer. By changing the composition, cross-sectional shapes, or
dimensions of the fibers throughout the spinning process, it is
possible to provide solids wherein the different layers are
comprised of different compositions, cross-sectional shapes, or
thicknesses. Several means to achieve this alignment are described
in Baker and Mauck, "The effect of nanofiber alignment on the
maturation of engineered meniscus constructs," Biomaterials, 28
(2007) 1967-1977, which is incorporated in its entirety by
reference for this purpose.
[0084] In some embodiments, at least one of the first layer and the
second layer has a population of cells disposed thereon. These
populations of cells can exist within the composition as
homogeneous or heterogeneous mixtures, and as at least one gradient
either across a radial and axial/longitudinal distance, or both.
These gradients can be continuous or step-wise, as with the other
components, as determined by the processing parameters.
[0085] Techniques for seeding cells onto or into a scaffold are
well known in the art, and include, without being limited to,
static seeding, filtration seeding and centrifugation seeding. See,
e.g., Baker and Mauck, "The effect of nanofiber alignment on the
maturation of engineered meniscus constructs," Biomaterials, 28
(2007) 1967-1977, which is incorporated in its entirety by
reference for this purpose. Static seeding includes incubation of a
cell-medium suspension in the presence of the scaffold under static
conditions and results in non-uniform cell distribution (depending
on the volume of the cell suspension); filtration seeding results
in a more uniform cell distribution; and centrifugation seeding is
an efficient and brief seeding method (see for example,
EP19980203774).
[0086] The cells may be seeded directly onto the scaffold, or
alternatively, the cells may be mixed with a gel which is then
absorbed onto the interior and exterior surfaces of the scaffold
and which may fill some of the pores of the scaffold. Capillary
forces will retain the gel on the scaffold before hardening, or the
gel may be allowed to harden on the scaffold to become more
self-supporting. Alternatively, the cells may be combined with a
cell support substrate in the form of a gel optionally including
extracellular matrix components.
[0087] The cells which can be used according to the teachings of
the present disclosure may comprise non-autologous cells or
non-autologous cells (e.g. allogeneic cells or xenogeneic cells),
such as from human cadavers, human donors or xenogeneic (e.g.
porcine) donors.
[0088] The cells may comprise a heterogeneous population of cells
or alternatively the cells may comprise a homogeneous population of
cells. Such cells can be, for example, stem cells (such as adipose
derived stem cells, embryonic stem cells, bone marrow stem cells,
cord blood cells, mesenchymal stem cells, adult tissue stem cells,
induced pluripotential stem cells,), progenitor cells (e.g.
progenitor bone cells), or differentiated cellarrays such as
chondrocytes, meniscal fibrochondrocytes, osteoblasts, osteoclasts,
osteocytes, connective tissue cells (e.g., fibrocytes, fibroblasts,
tenocytes, and adipose cells), endothelial and epithelial cells, or
mixtures thereof.
[0089] As used herein, the phrase "stem cell" refers to cells which
are capable of differentiating into other cell types having a
particular, specialized function (i.e., "fully differentiated"
cells) or remaining in an undifferentiated state hereinafter
"pluripotent stem cells".
[0090] Furthermore, such cells may be of autologous origin or
non-autologous origin, such as postpartum-derived cells (as
described in U.S. application Ser. Nos. 10/887,012 and 10/887,446).
Cells may be selected according to the tissue being generated.
[0091] It will be apparent that the disclosure has applications
beyond medical tissue engineering. For example, certain embodiments
comprise industrial applications, including dimensionally shaped
filters, textile, composites, catalyst scaffolds, electrolytic cell
diaphragms, battery separators, and fuel cell components.
[0092] Certain other embodiments provide that the fibrous material
also comprises a catalyst, including biological catalysts, for
example, but not limited to, enzymes, anti-microbials, or
anti-fungals. The means for incorporating these catalysts onto or
within electrospun materials are well known in the art as being
analogous to those used to incorporate particles as described
above. The means for functionalizing, immobilizing and/or attaching
catalysts onto polymer materials are equally known to those skilled
in the art. The first and second layers may have different
populations of cells disposed thereon. As an example, the first
layer may include stem cells, and the second layer may include
connective tissue cells.
[0093] The present disclosure also provides methods. These methods
include irradiating a first fibrous layer comprising a first
population of polymeric fibers having a first population of
nanoscale bodies disposed within, the irradiating being performed
so as to bond at least a portion of the first layer to a second
fibrous layer comprising a second population of polymeric fibers.
Without being bound to any single theory, one may consider the
irradiating as causing local heating at or around the nanoscale
bodies so as to bond the first and second fibrous layer together at
or near to the site of the irradiation. It should be understood
that the first and second layers may both contain populations of
nanoscale bodies.
[0094] Suitable nanoscale bodies are described elsewhere herein. As
explained above, a nanoscale body suitably has at least one
cross-sectional dimension in the range of from about 1 nm to about
100 nm, or even in the range of from about 10 nm to about 50
nm.
[0095] The irradiating may be effected by lasing, suitably by an
infrared laser. An exemplary laser is described elsewhere herein.
The user of ordinary skill in the art will be equipped to select a
laser of a suitable wavelength to effect the desired local
heating.
[0096] In some embodiments, a user may dispose the first population
of nanoscale bodies within a polymeric fluid so as to form a first
mixture and then electrospin the first population of polymeric
fibers from the first mixture. Additional information concerning
electronspinning is presented elsewhere herein. A user may further
dispose the second population of nanoscale bodies within a
polymeric fluid so as to form a second mixture and then form the
second population of polymeric fibers (e.g., via electrospinning)
from the second mixture.
[0097] The populations of polymeric fibers may be, as described
elsewhere herein, formed from one or more material that is natural,
synthetic, biocompatible, biodegradable, non-biodegradable,
biosorbable, or some combination thereof.
[0098] The first population of fibers, the second population of
fibers, or both, has an average cross-sectional dimension in the
range of from about 10 nm to about 10,000 nm, or in the range of
from about 100 nm to about 1000 nm, or from about 200 nm to about
500 nm.
[0099] The first population of polymeric fibers, the second
population of polymeric fibers, or both, may be characterized as
being an anisotropic alignment of fibers. The fiber alignment may
be linear, but may also be arcuate, as described elsewhere herein.
In some embodiments, at least some of the fibers in the first layer
have a different alignment than some of the fibers of the second
layer. In some embodiments, at least some of the fibers in the
second layer are oriented essentially perpendicular to at least
some of the fibers of the first layer.
[0100] FIG. 9 presents exemplary process conditions for a fibrous
layer that contains nanoscale bodies. As shown in the figure,
nanoscale gold nanorods may be disposed within a polymer.
Application of radiation (e.g., laser) may then effect local
heating so as to raise the temperature of the polymer at the site
of the irradiation to around or above the melting temperature of
the polymer, thus allowing the user to melt together adjacent
layers of material. Further information is provided in FIG. 10,
which figure presents an exemplary scheme for forming a laminate.
As shown in the figure, gold nanorods (NRs) were added to
poly-caprolactone (PCL) polymer, which polymer was then electrospun
into layers. The layers were then irradiated with a laser at 1 W of
power at 770 nm wavelength for about 45 seconds at a distance of
about 5 mm with a focusing filter in place. The irradiation
resulted in local fusion between adjacent layers.
[0101] Also provided are methods. These methods include
electrospinning, from a polymeric fluid, a first population of
polymeric fibers onto a first rotating surface of a mandrel, the
electrospinning being performed such that at least a portion of the
first population of polymeric fibers is aligned on the first
surface in an arcuate fashion.
[0102] In some embodiments, a spinneret containing the polymeric
fluid is oriented essentially perpendicular to the plane of the
first rotating surface of the mandrel. The spinneret may, in some
embodiments, be oriented essentially parallel to an axis about
which the first rotating surface of the mandrel rotates. The
spinneret need not be oriented exactly parallel to the mandrel's
axis of rotation, and the spinnarent need not be aligned so that it
is in register with the axis of rotation. For example, in the case
of a disc-shaped mandrel, the spinneret may be aligned with a point
that is at a radial distance from the center of the disc. The
spinneret may, of course, be aligned with the center of the
disc.
[0103] In some preferred embodiments, such as the embodiment shown
in FIG. 19A, the spinneret dispenses polymer so as to electrospin a
fiber onto a rotating mandrel. The mandrel suitably has an angular
velocity, which in turn allows the fibers to align in a
circumferential direction. As explained elsewhere herein, fibrous
materials (which may be termed "scaffolds") formed according to
these methods may have a spatially varying macrostructure. The
mandrel is suitably circular, although circular mandrels are not
necessary. The surface of the mandrel also need not be
perpendicular to the spinneret. Fibrous materials may be formed in
an arrayed fashion, in which multiple mandrels rotate as multiple
spinnarets dispense polymer onto the mandrels so as to
simultaneously form fibrous layers on the mandrels.
[0104] The polymeric fibers may be formed of a polymer that is
natural, synthetic, biocompatible, biodegradable,
non-biodegradable, biosorbable, or any combination thereof, as
described elsewhere herein. Fibers that comprise polycaprolactone
are considered especially suitable.
[0105] A user may also deposit a cell or even a population of cells
onto the electrospun fiber. It should be understood that this
deposition is not limited to applying the cells to a surface of the
fiber, as a user may contact or otherwise immerse the fiber into a
cell-containing medium. Stem cells and other cells (including those
cells described elsewhere herein) are all considered suitable for
this application.
[0106] At least a portion of the mandrel surface onto which the
fiber is electrospun suitably has a linear velocity during
electrospinning of between about 8 m/s and about 12 m/s; linear
velocities of about 10 m/s are considered especially suitable.
[0107] The polymeric fiber may be electrospun so as to form a body
having at least one cross-sectional dimension in the range of from
about 10 micrometers to about 1 cm, or from about 50 micrometers to
about 0.5 cm. Bodies formed according to these disclosed methods
are suitably fibrous layers in form. The fibrous layers may then be
cut to conform to a shape that a user desires.
[0108] In some embodiments, the rotating surface of the mandrel
comprises a first conductive region and a second conductive region
separated by an insulating region disposed there between. Such a
pattern may be characterized as being a target or bulls-eye
pattern. These patters may be used to give rise to a plurality of
polymeric fibers that are aligned radially relative to an axis
about which the first rotating surface of the mandrel rotates.
These radially aligned fibers may then be disposed adjacent to
fibers that have a arcuate (e.g, circumferential) alignment, with
the radially aligned fibers acting as "tie fibers" relative to the
arcuately aligned fibers.
[0109] The present disclosure also provides compositions. These
compositions suitably include a first layer comprising a first
population of polymeric fibers, the first population of polymeric
fibers having an anisotropic alignment that varies spatially within
the layer. An exemplary material is shown in FIG. 19B and FIG. 19C,
which figures show spatial variation of fiber alignment within a
fibrous material. At least a portion of the first population of
polymeric fibers has an arcuate alignment; fibers having a circular
or circumferential alignment are considered especially suitable. At
least a portion of the first population of fibers suitably has an
average diameter in the range of from 10 nm to about 10
micrometers. The population of fibers may contain a population of
nanoscale bodies; suitable such bodies are described elsewhere
herein.
[0110] The disclosed compositions may also include a population of
cells contacting the first population of fibers. The cells may be
stem cells, connective tissue cells, or other cells described
elsewhere herein. The polymeric fibers may be formed from materials
that are natural, synthetic, biocompatible, biodegradable,
non-biodegradable, and/or biosorbable, and unless specifically
restricted to one or more of these categories, the fibers may
comprise materials from any one of these categories.
[0111] Compositions according to this disclosure may include a
second fibrous layer, the second fibrous layer comprising a second
population of fibers. The second fibrous layer is suitably bonded
to the first fibrous layer. The second fibrous layer may comprise a
second population of fibers having an average diameter in the range
of from 10 nm to about 10 micrometers. The second population of
fibers may be aligned essentially parallel to one another. In some
embodiments, the second population of fibers has an arcuate
alignment. The alignments of the first and second populations of
fibers may be different; i.e., the second population of fibers is
aligned in a direction that differs from the alignment of the first
population of fibers. The second population of fibers may include a
population of nanoscale bodies disposed within. Suitable such
bodies are described elsewhere herein. The compositions may include
a porogenic material. The porogenic material may be disposed within
the first layer, the second layer, or even both.
[0112] The porogenic materials may be used, as one example, to give
rise to a material that includes anisotropically aligned fibers and
also includes sufficient void space so allow for cell seeding or
even cell ingrowth. As one example, a use may form a fibrous layer
from two electrospun polymer materials--a first material that is
comparatively fast to degrade under certain conditions, and a
second material that is slower to degrade under conditions that
degrade the first material. The user may form the desired layer
from these two materials; by dispensing both materials from the
same spinneret (or even from different spinnarets) and applying the
methods disclosed herein, the user can create a fibrous layer that
includes fibers of both materials, the fibers all sharing the same
alignment. The user may then expose the fibrous material to
conditions that degrade the first material, the degradation of that
first material in turn giving rise to void spaces within the
fibrous layer. Cells may then be seeded into or grow into the void
spaces left behind by the now-degraded first material.
[0113] The user may then, depending on their needs, install the
seeded implant into a subject. The second material of the implant
may be chosen to degrade over time under physiological conditions
so as to allow the subject's own cells to take the place of the
second material as that material degrades, while still providing
rigidity to the implant before degradation. Alternatively, the
second material may be one that does not degrade under
physiological conditions.
[0114] The present disclosure also provides biocompatible implants.
These implants suitably include a quantity of a composition
described herein, e.g., a composition that has a first layer
comprising a first population of polymeric fibers, the first
population of polymeric fibers having an anisotropic alignment that
varies spatially within the layer. The quantity of material may be
shaped so as to approximate at least a portion of a knee meniscus,
at least a portion of an annulus fibrosis, or other fibrous tissue.
Examplary annulus fibrosis and meniscus tissues are shown in FIG. 6
and FIG. 8, which figures illustrate the fibrous, laminate
structure within the tissues.
[0115] The present disclosure also provides methods, which method
include seeding a composition described herein with a population of
cells. The cells may be autologous, non-autologous, or both. The
user may then implant the seeded composition into a subject.
[0116] Non-limiting FIG. 18 is illustrative of these methods. As
shown in the figure, a user may form one or more fibrous layers,
which layers include fibers having an anisotropic alignment, and
which fibers may also include nanoscale bodies. The user may then
cut the layers to shape. As shown in FIG. 18, the layers may be cut
into crescent shapes and then fused to one another by application
of laser radiation to the layers, which radiation in turn effects
local heating and bonds the layers together. The application of
radiation may be performed in an arrayed fashion, where radiation
is simultaneously applied to the fibrous layers at a variety of
locations, e.g., by an array of lasers. Alternatively, radiation
may be applied sequentially to a variety of locations.
[0117] By cutting the layers to different shapes and laying the
layers atop one another, the user may then "build up" a
three-dimensional body that has an anatomic form and that also
features arcuately aligned fibers within, the alignment of the
fibers replicating the alignment of the fibers in
naturally-occurring tissue. The user may seed the layers with cells
before, during, or even after the layers are bonded to one another
so as to form the anatomic construct.
EXEMPLARY EMBODIMENTS
[0118] The following results are illustrative only and do not limit
the scope of the disclosure. Exemplary processing and testing
schemes are outlined in FIG. 10 and FIG. 11.
[0119] Thermal Bonding
[0120] Gold nanorods (NR) were fabricated. NR were suspended in a
solution of poly(.epsilon.-caprolactone) (PCL) in 1:1 THF:DMF at
varying concentrations prior to electrospinning into thin
(.about.500 .mu.m thick) fibrous scaffolds. Optimization of NR
concentration was carried out on 10 mm.times.10 mm strips using a
handheld IR laser (Chameleon Ti: sapphire laser, 770 nms, 1.0 W)
distanced .about.5 mm from the sample. IR light was focused through
a .about.1 mm diameter filter, and exposure time was set at
.about.45 seconds. Nanorod-containing scaffolds (NRS) and PCL-alone
scaffolds (PCL) were sputter coated and viewed under SEM. Fiber
size was determined using ImageJ. For mechanical testing, scaffolds
were extended to failure at 0.1%/sec using an Instron 5848.
Laminates were formed by overlaying 20.times.10 mm scaffold strips,
with a 10 mm overlap region. Fusion of layers was accomplished
(with one or two weld points) via a heated probe or via IR exposure
as above. Laminates were extended to failure at a rate of 0.1%/sec,
and maximum load of the interface recorded. Bovine MSC viability
and morphology was assessed on NRS with and without prior IR
exposure via Live/Dead and Actin-DAPI staining through day 7.
Statistical significance was determined by ANOVA with Tukey's
post-hoc tests (p<0.05).
[0121] Inclusion of gold nanorods within PCL fibers (FIG. 1A) had
no effect on fiber morphology, as shown in FIG. 1C and FIG. 1D, as
well as by FIGS. 12, 13, and 17. Cell viability and shape on the
NRS appeared normal, with cells elongated in the fiber direction
(FIG. 1B). Further, NRS modulus did not differ from PCL (FIG. 1F),
though a decrease in yield stress was observed (p<0.05) (FIG.
2G). Heat generation was essentially proportional to NR
concentration; the minimum NR concentration required to melt fibers
locally was 6.3.times.10.sup.-14 mol NR/mL.
[0122] Laminates were readily formed with IR exposure of NRS, with
both one and two weld points (FIG. 2). For both annealing methods,
increasing weld number increased interface strength (FIG. 2), with
spot welding via heated probe having .about.2-3-fold higher
strength than laser-mediated assembly (p<0.05). Outside of the
welds, cell number and morphology were similar for both methods
(not shown). It should be understood that a user may irradiate
adjacent layers at one, two, or more locations.
[0123] NRs incorporated into electrospun PCL fibers did not
substantially alter fiber morphology or scaffold mechanical
properties, as shown in FIG. 15. As shown by FIG. 14,
NR-irradiation welding retained more fibrous structure than did
welding layers using a heated probe. Local heating of gold NRs
could be achieved with focused application of IR light and by
varying the concentration of NRs within the spinning solution.
Inclusion of NRs within the fibers did not appear to alter cell
viability or morphology after one week of culture. Consistent with
the previous annealing method (heated probe), cell morphology was
only perturbed within the weld area, suggesting normal fiber
architecture outside the exposure site. (see FIG. 14) Welds were
capable of holding layers in apposition for a long enough period of
time for cell colonization and matrix deposition to occur. As shown
in FIG. 16, nanorod-mediated infrared welding produced a stable
interface, which interface had increasing mechanical properties
with increasing weld number.
[0124] Moreover, as the NR-mediated method does not require
physical contact with the scaffold, local compression is not a
concern. This process is applicable to the assembly of anatomic
laminate structures for engineering complex tissues such as the
knee meniscus and annulus fibrosis.
[0125] Electrospun Scaffolds
[0126] Scaffold Fabrication:
[0127] Electrospinning of poly(-caprolactone) (PCL) nanofibers was
carried out to form linearly aligned fiber scaffolds. Additionally,
circularly aligned (CircAl) PCL scaffolds were formed by modifying
the collection apparatus such that the polymer was centered above
and perpendicular to the plane of a grounded rotating aluminum
collecting plate (FIG. 5A).
[0128] Fiber Alignment:
[0129] To quantify the alignment of fibers, thin films were
collected for .about.5 minutes on glass slides (n=6/scaffold type)
and viewed by light microcopy (Eclipse 90i, Nikon Instruments). For
CircAl fibers, fibers were collected at a radius of 3 cm on the
mandrel. Images were collected at 4.times. magnification over a 20
mm by 10 mm area using a motorized stage and an image stitching
program (NIS Elements software, version 3.22). Fiber alignment of
individual images was quantified via a custom MATLAB script
utilizing Fast Fourier Transform analysis. Mean fiber alignment was
determined as a function of spatial position on the slide (FIG.
3A). For statistical analysis, Pearson correlation coefficients
were calculated between mean fiber alignment and spatial location,
with significance set at p<0.05. FIG. 3B illustrates the
circumferential alignment of fibers as a function of the fibers'
distance from the center of the rotating mandrel on which the
fibrous body was formed.
[0130] Cellular Interactions:
[0131] Additional scaffolds (30 mm.times.10 mm, n=3) were utilized
to assess cellular morphology. Juvenile bovine mesenchymal stem
cells were harvested and cultured up to P2 as previously described
(5). Cells (80,000) were deposited on each scaffold, cultured for 5
days, and fixed in 4% paraformaldehyde. Cell nuclei and F-actin
were visualized with 4',6-diamidino-2-phenylindole (DAPI) and
phalloidin-Alexa488 (Invitrogen), respectively, and imaged on a
fluorescent microscope (Eclipse 90i, Nikon Instruments).
[0132] Mechanical Testing:
[0133] Oriented scaffolds of .about.0.7-0.8 mm thickness were
formed (n=7/scaffold type). For the CircAl scaffolds, specimens
were excised from oriented mats at two radial positions (3 cm and 5
cm). All scaffolds were excised at two lengths (30 and 60 mm) with
a 3 mm width (FIG. 5A, n=7/group). The cross-sectional area of the
samples was calculated using a custom laser-based device. Samples
were then sputter coated with Verhoff's stain and placed in
custom-made grips. Clamp-to-clamp length was set at 15 mm and 45 mm
for the short and long strips, respectively. Mechanical evaluation
was performed using a materials testing machine (Instron, model
5848) in a saline bath at 37.degree. C. Samples were preloaded to
0.5N, subjected to 15 cycles of preconditioning from 0-3%, and then
extended to failure at a rate of 0.5%/sec. Images were acquired at
a rate of 2 frames per second. Lagrangian strain in an area near
the scaffold center and an area of equal size near the grips were
computed using Vic2D (Correlated Solutions). Tensile modulus for
each region was calculated as the slope of the stress-strain curve
between 1-3% strain. For statistical analysis within each scaffold
type, a two-way repeated measures ANOVA was performed, followed by
paired t-tests to determine statistical differences between
individual groups. Overall statistical significance was maintained
at p<0.05.
[0134] By collecting fibers on a rapidly revolving surface,
organized nanofibrous scaffolds with pronounced curvature could be
formed (FIG. 3). Fiber orientation varied linearly across a 20 mm
distance, ranging from .about.103.degree. to .about.77.degree. at a
radius of 3 cm. Statistical analysis confirmed this relationship
with a Pearson correlation coefficient of -0.963 (p<0.05).
Alternatively, linearly aligned (LinAl) fibers were centered at
90.degree. and did not vary with position (R=-0.111,
p>0.05).
[0135] When cells were seeded on CircAl and LinAl scaffolds,
cytoskeletal and nuclear morphology was similar (FIG. 4). The
cells, however, followed the local directionality of the scaffold,
with alterations in cellular alignment on a macroscopic scale
clearly observed on the CircAl scaffolds (FIG. 4B). As seen in FIG.
3A, the cellular alignment was essentially unchanged along the
length of a linearly-aligned fiber
[0136] Mechanical analysis of the CircAl scaffolds revealed
significant interactions between scaffold length and region within
the scaffold (p<0.05, FIG. 5B). As such, comparisons between
groups were done on an individual basis. For short specimens, no
significant differences were found in the modulus between the
center and edge regions of the CircAl specimens (p>0.01).
Conversely, for long specimens, the modulus was 32-36% lower near
the edge of the scaffolds compared to the center (p<0.01). In
terms of scaffold length, the modulus of the central region of the
CircAl specimens was similar between short and long specimens
(p>0.01); however, the modulus of the edge region for the short
CircAl specimens was 68-90% higher than the respective long
specimens (p<0.01). No statistically significant differences
could be detected between LinAl specimens as a result of varying
specimen length or region within the scaffolds (p>0.05). These
differences in modulus are further highlighted by a graphical
depiction of the Lagrangian strain along the direction of loading
(FIG. 5C).
[0137] Thus, the disclosed methods allow for the construction of
electrospun nanofibrous scaffolds with a spatially varying
macroscopic fiber orientation. This orientation is similar to the
meniscus. On the microscopic scale, these fibers were locally
aligned, allowing alignment of MSCs (mesenchymal stem cells)
similar to linearly aligned scaffolds. The CircAl fibers followed a
pattern roughly parallel to the circumferential direction of the
disc, creating a linear gradient in fiber angle along the length of
excised scaffolds and resulting in MSCs with a similar change in
morphology over a macroscopic scale, in support of our
hypothesis.
[0138] The CircAl fiber scaffolds behaved similarly to linearly
aligned scaffolds over a short spatial domain, but varied
considerably as specimen length increased. Without being bound to
any single theory, increasing the length of specimens for tensile
testing may increase the frequency of CircAl fibers that enter
and/or exit before reaching the clamped regions, resulting in
greater regional differences within the scaffolds (i.e. decrease in
modulus near the edge). These layers may be combined as shown in
FIG. 18 to generate anatomic 3D implants that recreate the
macroscopic and microscopic features of the native tissue. The
organized nanofibrous scaffolds generated herein replicate the
macroscopic curvature of the native tissue, and can direct the
formation of an anatomic construct with direction dependent
mechanical properties that vary across a large anatomic
expanse.
* * * * *